Reabsorption and cellular handling of glomerular filtered vitamins, peptides, and hormones in the proximal tubule are essential, but thus far, poorly elucidated processes. The multiligand receptor ...megalin, initially described as a Heymann nephritis antigen and later identified as a member of the LDL receptor gene family, mediates reabsorption of several molecules, such as transcobalamin-vitamin B12 and albumin, in the proximal tubule. Consequently, a differentiated cell line of proximal tubular origin expressing megalin is an important requisite for examination of the above-mentioned processes. This study shows, using electron microscopy, that the cell line LLC-PK1, originating from the proximal tubule, maintained differentiated morphology and had a well developed endocytotic apparatus. Furthermore, by immunoblotting and immunohisto- and cytochemistry, megalin was identified in the endocytotic compartments of these cells. Megalin was situated mainly in the endosomes and in the dense apical tubules, but it was also identified in coated pits and in the brush border. The ability of megalin to mediate internalization and degradation of labeled receptor-associated protein (RAP) in a RAP-inhibitable manner was demonstrated. By autoradiography, the endocytosed, iodinated RAP was located in endosomes and lysosomes in the apical part of the cells. Moreover, the LLC-PK1 cells assembled in a monolayer with a hindrance toward diffusion of labeled mannitol, inulin, and dextran at a satisfactory level for the study of proximal tubule handling of smaller proteins. This study reveals a proximal tubule cell line expressing megalin in a functional manner well suited for binding, uptake, and transcellular transport studies.
Intravital microscopy theoretically provides the optimal conditions for studying specific organ functions. However, the application of microscopy in intact organs in vivo has been limited so far due ...to technical difficulties. The purpose of this study was to establish a method of in vivo confocal laser scanning microscopy (CLSM) for the study of endocytosis in proximal tubules of intact kidney.
The left kidney of rats placed on a modified microscope stage was exposed and stabilized in a thermostatically controlled cup. The stage was then attached to an upright confocal microscope. Surface proximal tubules were microinfused with fluorescent albumin or transferrin. Single or time-series images of microinfused proximal tubules were recorded in reflection and/or fluorescence mode.
The stability of the kidney and the resolution of images were sufficient to visualize intracellular vesicles. Albumin and transferrin were initially observed at the brush border, then later internalized by proximal tubules and accumulated in lysosomes over a time period of 15 min. Furthermore, fusion of vesicles was observed in time-lapse images.
These results show that in vivo CLSM in intact kidney may be an excellent method to evaluate proximal tubular endocytosis and ligand trafficking.
Folate derivatives are filtered in the glomeruli and reabsorbed within the nephron. The amount filtered largely exceeds the minimum daily requirements. Thus folate reabsorbed within the kidney must ...be returned to the circulation. To establish whether renal proximal tubule can accomplish this by transport, of 3Hfolate across the cell, microperfusion of rabbit, proximal tubule with 3Hfolate and 14Cinulin was performed. Transtubular transport of 3Hfolate was 5 +/- 1% (0.25 +/- 0.07 fmol/min) of perfused amount/mm tubule and remained constant during a 2-h perfusion period. An accumulation of 15 +/- 4% (0.8 +/- 0.3 fmol/min) of perfused amount/mm tubule was observed during the same period. Furthermore, to determine whether endocytosis may be involved in the initial process of folate uptake in proximal tubule cells, we performed light microscopy autoradiography on cryosections of rat kidney cortex incubated with 3Hfolate. Folate binding sites were located apically as well as intracellularly similar to the location of 3Hfolate when injected into the abdominal aorta and visualized by light microscopy autoradiography. Thus folate binding sites as well as internalized folate is localized both apically and intracellularly. Micropuncture of rat proximal tubules with folate-coupled collodial gold particles showed significantly increased endocytosis of folate gold when evaluated quantitatively and compared with controls injected with noncoupled gold particles (0.22 +/- 0.08 vs. 0.03 +/- 0.01 gold particles/micron 2 tubule cell). The results show that kidney proximal tubule cells are capable of transcellular transport of 3Hfolate with limited capacity. Folate gold particle uptake suggests that folate can be internalized by endocytosis.
The renal proximal tubule exhibits a very extensive apical endocytic apparatus consisting of an elaborate network of coated pits and small coated and noncoated endosomes. In addition, the cells ...contain a large number of late endosomes/prelysosomes, lysosomes, and so-called dense apical tubules involved in receptor recycling from the endosomes to the apical plasma membrane. This endocytic apparatus is involved in the reabsorption of molecules filtered in the glomeruli. The process is very effective as demonstrated by the fact that although several grams of protein are filtered daily in the human glomeruli, human urine is virtually devoid of proteins under physiological conditions. Several key receptors appear to be involved in this function, which serves not only to conserve protein as such for the organism but also to reabsorb vital substances such as different vitamins in complex with their binding proteins. Recent research has established megalin, a 600-kDa protein belonging to the LDL receptor family, as probably the most important receptor in this process in the proximal tubule mediating endocytosis of a large variety of ligands and therefore classifying it as a scavenger receptor. More specific receptors like the folate receptor, IGF-II/Man-6-P receptor, and gp280/IFR, identical to the intrinsic factor receptor, are also functioning in the apical endocytic pathway of renal proximal tubules. A better understanding of these receptors will give us new insight into these very important processes for the organism.
The multiligand, endocytic receptors megalin and cubilin are colocalized in the renal proximal tubule. They are heavily expressed in the apical endocytic apparatus.
Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in renal ...proximal tubules has not been clarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were captured from solubilized renal brush-border membranes by affinity chromatography using myoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with Kd values of 2.0 and 3 µM, respectively, as evaluated by surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced (Kd = 5 µM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renal brush-border membranes (Ki 2 µM). Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin by cultured yolk sac BN-16 cells. The significance of megalin- and cubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated by use of kidney-specific megalin knockout mice. Injected myoglobin was extensively reabsorbed by megalin-expressing proximal tubular cells, whereas there was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in the renal proximal tubule involving the endocytic receptors megalin and cubilin. Identification of the receptors for tubular uptake of myoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.
High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of 3Hfolate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has ...not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.
The transcobalamin-vitamin B(12) complex is responsible for the transport of B(12) from plasma and into the tissues. The complex is filtered in the renal glomeruli and is a high-affinity ligand for ...the endocytic receptor megalin expressed in the proximal tubule. This study shows by the use of the proximal tubule LLC-PK1 cell line that transcobalamin-B(12) is internalized by megalin-mediated endocytosis. After endocytosis and accumulation in endosomes, transcobalamin is degraded and the B(12) molecule is released from the cells in complex with newly synthesized proteins. The release is polarized in such a way that vitamin in the apical medium is bound to proteins with the size of haptocorrin, whereas the B(12) released at the basolateral side is complexed to two different proteins with the sizes of transcobalamin and haptocorrin. Furthermore, transcobalamin mRNA was identified by reverse transcription-PCR in LLC-PK1 cells and human and pig kidney, whereas haptocorrin mRNA was identified only in LLC-PK1 cells. The results strongly suggest that megalin located in the proximal tubule cells is important for receptor-mediated tubular reabsorption followed by transcellular transport and release of vitamin B(12) complexed to newly synthesized carrier proteins. This mechanism is likely to play a significant role in the maintenance of B(12) homeostasis by returning filtered B(12) to the pool of circulating vitamin.
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